As a method for directly detecting a specific reaction without labels, a method for detecting a specific binding based on changed refractive indexes via surface plasmon resonance (SPR) has been put to practical use. This enables specific detection of low-molecular-weight molecules. Thus, such method has been applied to drug screening or used for other applications.
For example, JP Patent Publication (kokai) No. 2002-148187 A discloses a surface plasmon resonance enzyme sensor chip used for a surface plasmon resonance apparatus, which comprises an optically transparent substrate and a thin metal layer of gold or silver formed thereon, the metal thin film being modified with a membrane that undergoes an electron transfer reaction with both the thin metal layer and an enzyme. Also, JP Patent Publication (kokai) No. 2003-232725 A discloses a sensor for analyzing chemical reactions which comprises; a prism; a thin metal layer thereon; a sensor chip comprising a flat micro-fluid channel having a thickness of 30 μm or smaller in which a sample flows while being in direct contact with the surface of the thin metal layer; a light source means for irradiating the back surface of the thin metal layer via total internal reflection through the prism; and a means for measurement allowing the measurement of the spatial distribution of the refractive index of the light reflected from the sensor chip with the elapse of time. A sample is allowed to flow through the flat micro-fluid channel, the spatial distribution of the refractive index resulting from physical or chemical reactions of the sample is measured with the elapse of time, and the reaction rate of physical or chemical reaction of the sample is measured based on the results of measurement.
Many target proteins of drug screening are functional proteins, such as enzymes. In the field of drug discovery, accordingly, it is important to obtain a compound that would influence the activity of functional proteins. When only the binding of a compound to a protein is detected based on SPR signals, however, whether or not its binding is binding to an active site of the protein, binding to another site, or binding to an inactivated protein, could not be determined.
If binding of a compound to a functional protein is detected and a reaction product derived from the activity of the functional protein can then be assayed again, accordingly, whether or not the initial binding of a compound would affect the activity can be determined.
JP Patent Publication (kokai) No. 2005-24483 A discloses a biosensor according to a different concept, which is a biosensor for detecting a molecule associated with specific binding of a biomolecule, which comprises: (i) a reaction region where a) a specific binding reaction and b) an enzyme reaction are performed; (ii) a detection region where a product of an oxidation-reduction reaction resulting from the reactions a) and b) is reacted with a membrane of an oxido-reducing substance; and (iii) an assay region where changes in conditions of the membrane of an oxido-reducing substance resulting from the reaction with a product of an oxidation-reduction reaction are assayed to determine changes in dielectric constant. The invention of JP Patent Publication (kokai) No. 2005-24483 A is intended to provide a biosensor that can provide amplification effects via an enzyme in a micro-fluid channel. According to a highly-sensitive assay technique employing a specific binding reaction and enzyme amplification, a membrane that captures a product of an enzyme reaction at a site downstream of a micro-fluid channel is provided, and the progress of such capture is assayed by measuring the dielectric constant of the membrane, so that the effects of enzyme amplification can be realized without restriction regarding the volume of the fluid channel, even when a substrate continuously flows through the micro-fluid channel. Thus, a lower limit of detection in terms of concentration can be achieved. With the use of such biosensor, however, reaction products are limited to products of oxidation-reduction reactions. Accordingly, such sensor cannot be applied to the evaluation of the binding of a compound to any type of functional protein.